Pancreatitis is a potentially fatal disease of exocrine pancreas the pathogenesis of which remains obscure and for which no specific treatment has been developed. Alcohol abuse is a major etiologic factor for pancreatitis; however, the mechanisms by which alcohol predisposes to this disease remain elusive. Here we propose a novel hypothesis for the pathogenesis of alcoholic pancreatitis stating that a combination of two events is critical for pancreatitis: 1) induction of autophagy and 2) impaired lysosomal function which makes autophagy defective. Autophagy (a.k.a. macroautophagy) is the main cellular degradative, lysosome-driven process. Our recent study has revealed a profound impairment of autophagy in experimental models of nonalcoholic acute pancreatitis. We found that autophagy is activated but its progression/resolution is impaired. Autophagy impairment is caused by lysosomal dysfunction a prominent manifestation of which is defective processing/maturation of cathepsins, major lysosomal hydrolases. Further, our findings revealed that impaired autophagy mediates 2 key pathologic responses of pancreatitis: acinar cell vacuolation and intra- acinar trypsinogen activation. Our results indicate that ethanol feeding alone causes lysosomal dysfunction in pancreas similar to that we found in nonalcoholic pancreatitis. However, ethanol per se does not induce pancreatitis because it does not activate autophagy. Thus, in conditions of basal unstimulated autophagy, the consequences of ethanol- induced lysosomal dysfunction are limited. However, a combination of ethanol feeding, which causes lysosomal dysfunction, and stresses that induce autophagy leads to defective autophagy and thus pancreatitis. Our results suggest that this is the mechanism through which ethanol feeding sensitizes rats or mice to pancreatitis induced by low-dose cerulein (CR; a CCK-8 analog) that by itself does not elicit pancreatitis. We propose that similar mechanism mediates pancreatitis induced by the combination of ethanol feeding and endotoxemia (i.e., LPS), the other available in vivo sensitization model of alcoholic pancreatitis. Our results indicate that ethanol causes dysregulation of endolysosomal trafficking and the Golgi, associated with defective processing and activity of cathepsins and decreased level of LAMP-2, a major lysosomal membrane protein. We further propose that manipulating the level of autophagy (by, respectively, inhibiting autophagy through Atg5 knockout or stimulating it through overexpression of Atg8/LC3, a critical autophagy mediator) ameliorates or worsens alcoholic pancreatitis. The Specific Aims for the project are: 1. Determine the effects of ethanol feeding alone and experimental alcoholic pancreatitis (i.e., ethanol feeding combined with low-dose CR or LPS) on lysosomal dysfunction in pancreas, and the role of LAMP-2 in these effects. 2. Determine the effects of ethanol feeding alone and experimental alcoholic pancreatitis on endolysosomal trafficking and the Golgi. 3. Determine the effects of ethanol feeding alone and experimental alcoholic pancreatitis on autophagy induction and progression, and the role of LAMP-2 in these effects. 4. Determine the effects of genetically inhibiting or stimulating autophagy on alcoholic pancreatitis. The proposed studies elucidate ethanol's effects on lysosomes, the Golgi, and autophagy in pancreas; suggest a new model for alcoholic pancreatitis; and indicate novel targets for therapeutic approaches to treat or mitigate this disease. Further, similar mechanisms involving ethanol-induced lysosomal dysfunction may mediate alcohol toxicity to other organs. PUBLIC HEALTH RELEVANCE: Pancreatitis is a serious and sometimes lethal disease of exocrine pancreas, the pathobiology of which is poorly understood, and specific treatments for which do not exist. Alcohol abuse is a major risk factor for pancreatitis; however, the mechanisms by which ethanol predisposes to pancreatitis remain unknown. In this project, we will test the hypothesis that a key mechanism is ethanol-induced damage to lysosomes, resulting in impaired autophagy in pancreas. Lysosome is the key cellular degradative organelle, and autophagy is the main process through which cellular components are digested in lysosomes. We will use mouse models to test our hypothesis. The results can lead to novel therapeutic approaches, targeting impaired lysosomal function in autophagy, to treat or mitigate alcoholic pancreatitis in humans. Further, similar mechanisms involving ethanol-induced lysosomal damage may mediate ethanol's toxicity to other organs.